The objectives of our research are to understand the mechanism of meiotic recombination initiation, and to determine how this process is coordinated with other events of meiotic prophase. In the previous period, we focused on yeast Spol 1 (the protein that makes the double-strand breaks (DSBs) that initiate meiotic recombination) and the proteins that interact with it. In the new project period, we propose to continue studying the mechanisms of DSB formation and repair. The specific aims are: 1. To determine the role of Ski8 in DSB formation. Ski8 interacts with Spo11 and associates with meiotic chromosomes. We will a) define the regions in Ski8 protein required for interaction with Spo 11, and b) ascertain the locations and genetic requirements of its association with chromosomes. 2. To characterize chromosomal interactions of meiosis-specific proteins required for DSB formation. We have found that the distribution of Rec 102 binding sites reflects the basic axis/loop structure of meiotic chromosomes, but that Rec 102 binding is not sufficient to account for the distribution of meiotic DSBs. To determine whether localization of other DSB proteins influences DSB distribution, we will characterize the association of Mer2, Mei4, and Rec114 with chromosomes. We have also found that Rec102 binds primarily to chromatin loops early in meiosis, but becomes more closely associated with chromosome axes later. We will test whether this spatial reorganization reflects association of Rec102 with the sites of ongoing recombination, and whether Rec102 (and/or Rec104) interact directly with chromosome axis components. 3. To determine the factors that dictate where DSB formation occurs. Missense spo11 mutations change the cleavage pattern within DSB hotspots, demonstrating that Spo11 itself contributes to the choice of cleavage site. Details of the altered DSB patterns have led us to propose that trans-acting "positioning" factors bound to the DNA in or near hotspots also contribute to the decision of where to cleave. We will test this hypothesis by asking which is more important for specifying cleavage position, the nucleotide sequence directly at the cleavage site or position within the hotspot. 4. To determine the mechanism of crossover homeostasis. We recently found that cells can maintain normal levels of crossing over even when DSB frequencies are reduced. We refer to this phenomenon as "crossover homeostasis." We will test whether crossover homeostasis and crossover interference are related.